1. Field of the Invention
The present invention relates to a technical field of specimen measurement for determining a specified trace component in the specimen utilizing, for example, an antigen-antibody reaction or hybridization of nucleic acid.
2. Related Background Art
The recent advances in the detection technologies of specified trace components in the specimen are playing a significant role in the field of clinical tests, for early diagnosis or discovery of various deseases. Since it was published in 1958 by S. A. Berson et al. that insulin was determined with radioactive iodine labelling, there have been established determinations of plasma protein such as IgE, IgG, CRP and microglobulin, tumor markers such as AFP, CEA and CA19-9, hormones such as TSH and T.sub.3, pharmaceutical substances in blood, virus as such as HBV and HIV, antibodies therefor, and nucleic acids such as DNA and RNA, with possibility for automated processing of plural specimens.
Most of these trace components are determined by immunoassays utilizing antigen-antibody reactions, or by methods utilizing nucleic acid-nucleic acid hybridization. In such analytical methods, for example, an antigen, an antibody or a single-chain nucleic acid capable of specifically binding with the object substance to be determined, is used as a probe, and is fixed on a solid surface such as small particles, beads or a reactor wall., and an antigen-antibody reaction or a nucleic acid hybridization with said object substance is effected. In such reaction there is employed a labelled antigen, a labelled antibody or a labelled nucleic acid, bearing a labelling substance of a high detection sensitivity such as an enzyme, a fluorescent substance or a light-emitting substance to detect an antigen-antibody complex or a double-chained nucleic acid, thereby determining the amount of the object substance to be determined.
FIG. 9 illustrates a typical example of fluoroimmunoassay (sandwich immunoassay) in which the object substance is an antigen. A first reagent is prepared, in which an antibody 24 constituting a first substance capable of specifically binding with an antigen 22 contained in the specimen, is fixed on carrier particle 21 in advance, and is mixed with a specimen such as serum to induce an antigen-antibody reaction with the antigen 22 therein (1st reaction). Then, a second reagent in which an antibody 23 capable of specifically binding with the antigen 22 is labelled with a fluorescent substance 25, is mixed to form a complex (2nd reaction). After 2nd reaction, an amount of the antigen 22 in the specimen is determined by the fluorescent measurement of the complex.
FIG. 10 shows an example of the fluorescent measurement of the above-mentioned sandwich immunoassay, utilizing so-called flow cytometry technology. A sample liquid containing complexes 31, 32 obtained through the reactions explained above is contained in a reaction liquid bath 38. It is to be noted that said sample liquid also contains various substances in the specimen, carrier particles, labelling substance or the like. The reaction liquid bath 38 is pressurized by a pump 39 to feed the sample liquid into a flow cell 35. In said flow cell, there flows sheath liquid 34 to generate a laminar flow due to sheath flow principle, whereby a plurality of complexes 31, 32 flow in a line. Said flow is irradiated by a laser light source 36 with a laser beam of a wavelength capable of exciting the labelling fluorescent substance. When the complex 31 is irradiated by the laser beam, a scattered light 37a is generated by the carrier particle, and a fluorescent light 37b is simultaneously emitted from the labelling substance 33a. However, when the labelling substance 33a, object substance 33b or carrier particle 33c each unreacted flows alone, the scattered light 37a and the fluorescent light 37b are not generated at the same time, or only at a very low intensity. Consequently, the complexes 31, 32 alone can be selectively measured, independently from other noise components, by collect the data only when the scattered light and the fluorescent light are generated at the same time and exceed a certain level.
FIG. 11 is a wave form chart indicating the signals of the scattered and fluorescent lights in the measurement shown in FIG. 10. In an ideal case shown in FIG. 11, the intensity of the forward scattered light is high when a complex reaches the irradiated area of the flow cell and is low except at said area, since a strong scattered light 51 is generated by the carrier particle. Therefore, a trigger signal 52 is generated when the intensity of the forward scattered light is beyond a predetermined level, and the intensity of the fluorescent light 53 is measured at the timing of said trigger signal 52, whereby the intensity of the fluorescent light from the complex can be selectively determined. If fluorescent light is generated, it can be judged the object substance is present and an antigen-antibody reaction or a nucleic acid hybridization reaction has been induced. On contrary, no fluorescent light is generated, it can be judged that such reaction has not been induced. The object substance can be determined by statistical processing of a multitude of measurements.
In practice, however, the ideal output as shown in FIG. 11 is not obtainable, since the increase in the light intensity is caused, as shown in FIG. 12, by factors other than the forward scattered light generated by the complexes themselves. As example of such factors there are floating substances present in the original reaction liquid and bubbles generated therein. In such a case, as shown in FIG. 12, the forward scattered light shows not only a peak 61 resulting from the complexes but also a peak 62 resulting from the floating substances or the bubbles. Consequently, in addition to a trigger signal 63 corresponding to the complexes, there is generated a false trigger signal 64 based on such floating substances or bubbles, so that the fluorescent intensity (zero-level) at that time is read. This causes a mistake as if there were carrier particles having not induced the antigen-antibody reaction or the nucleic acid hybridization reaction, significantly affecting adversely the precision of quantitative measurement.